This is a new proposal entitled Hepatic insulin resistance and metabolic disease that is based upon work conducted in our laboratory during the past 5 years with LDKO (hepatic-specific Irs1-/-Irs2-/-) and LTKO (hepatic-specific Irs1-/-Irs2-/-FoxO1-/-) mice. LDKO-mice display systemic metabolic dysregulation, including hepatic and peripheral insulin resistance, hyperglycemia, moderate hyperinsulinemia, and progressive NAFLD (nonalcoholic fatty liver disease). The etiology of NAFLD is rooted in insulin resistance, obesity, hyperlipidemia, and diabetes. As LTKO-mice display normal nutrient homeostasis, we posit that activated hepatic FoxO1, rather than the mere accumulation of hepatic triglyceride, promotes inflammation that progresses to life- threatening necrosis/apoptosis and cycles of regeneration that culminate with damage-characteristic of the progression of NAFLD to NASH (nonalcoholic steatohepatitis) and HCC (hepatocellular carcinoma). To investigate the underlying pathophysiology, we focus upon mitochondrial dysfunction that develops as a result of chronically activated FoxO1 that increases the expression of hundreds of genes, including hemeoxygenase- 1 (encoded by Hmox1) and cyclophilin D (encoded by Ppif). To interrogate the molecular mechanisms, we propose to use nanoparticle delivery of targeted siRNA to suppress the expression of hepatic FoxO1, Hmox1, or Ppif during initiation (10 weeks of age) and progression (10 months of age) of NAFLD. To enable this technology in our laboratory, we have formed a 'Consortium Agreement' with the Amiji group in the School of Pharmacy at Northeastern University. The mouse-based experiments will investigate the relation between FoxO1-mediated hepatic mitochondrial dysfunction and progressive NAFLD/NASH upon systemic inflammation and muscle insulin action that contributes to diabetes in the following Specific Aims: i) Investigate FoxO1-mediated mitochondrial dysfunction in LDKO-mice by modulating the expression of FoxO1, Hmox1 or Ppif to restore mitochondrial function and attenuate hepatic inflammation, NASH and its progression to HCC during persistent hepatic insulin resistance. ii) Investigate skeletal muscle insulin resistance in LDKO-mice to establish the relation between hepatic inflammation and dysregulated skeletal muscle insulin action and metabolism. iii) Quantify Ser/Thr-phosphorylation of IRS1 in muscle of LDKO-mice using a library of phosphosite-specific monoclonal antibodies before and after resolution of hepatic mitochondrial dysfunction and NAFLD by suppression of FoxO1, Hmox1 or Ppif. Since the LDKO-mice are uncomplicated by the dominant effects of hypothalamic-based obesity encountered with ob/ob-mice, our experiments focus squarely upon the relation between hepatic insulin resistance and NAFLD, and its progression to systemic insulin resistance and diabetes.